The primary objective of this research is the experimental observation and description of macromolecular dynamics in the protein calmodulin. These motions will be examined by intramolecular nonradiative energy transfer using conventional and frequency domain fluorometry. Transfer will take place between donors and acceptors which are strategically placed fluorescent, luminescent, and phosphorescent labels. Calmodulin, an important calcium regulatory protein, is known to respond to an intracellular increase of calcium by binding to four calcium ions, undergoing a conformational change which exposes a hydrophobic pocket, and in this "active" form, proceeding to bind and activate a host of different target proteins such as myosin light chain kinase, calcineurin, and phosphodiesterase. Energy transfer will be used to study the flexibility of the solution structure of calmodulin both in its complex with calcium and complexed to several of these target proteins. The synthesis and characterization of a calmodulin complex with a lanthanide ion bound to the N-terminal domain and a fluorescent dye molecule bound to Lys 148 on the C-terminal domain has been completed. In the energy transfer experiments proposed here, the luminescence quenching of the lanthanide donor by the rhodamine acceptor will first be used to measure the distance between two domains in the protein as a function of added calcium. The effects of target protein binding on the interdomain distance will be determined for an assortment of target proteins. In addition, the intrinsic flexibility of each of these structures can be measured by examining the heterogeneity in the intensity decay using frequency domain fluorometry as well as the thermal and quenching profiles of the energy transfer. Finally, metal and target protein induced changes in the fluorescence anisotropy of the dye (which is located adjacent to the hydrophobic pocket) should help to elucidate the molecular strategies by which calmodulin functions as the central intramolecular interpreter of the second messenger calcium. The availability of the cloned gene for calmodulin also makes possible additional studies of these conformational changes. By site specifically mutating Leu 39 and Val 91, two residues in calmodulin shown to exhibit enormous changes upon binding to target peptides, a clear picture of the dynamic response of the protein should emerge. Nearly 33 angstroms apart in the crystal structure, the methyl groups of these side chains are less than 5 angstroms apart in the ternary complex. Mutation of these residues to cysteine will allow for a multitude of cross-linking and derivatization experiments. Again, energy transfer studies such as those described above will be used to give insight into the dynamics of this dramatic conformational change.